In Escherichia coli (E. coli), programmed cell death is mediated through “addiction modules” consisting of two genes, one of which encodes a stable toxic protein (toxin) and the other encodes a short-lived antitoxin (Engelberg-Kulka and Glaser, Annu Rev Microbiol 53, 43-70 (1999)). The toxin and the antitoxin are coexpressed from an operon and interact with each other to form a stable complex and their expression is auto-regulated either by the toxin-antitoxin complex or by the antitoxin alone. When their co-expression is inhibited by stress conditions, for example, the antitoxin is degraded by proteases, enabling the toxin to act on its target. Such genetic systems for bacterial programmed cell death have been reported in a number of E. coli extrachromosomal elements for the so-called postsegregational killing effect (Tsuchimoto et al., J Bacteriol 170, 1461-6 (1988); Roberts and Helinski, J Bacteriol 174, 8119-32 (1992)). When bacteria lose the plasmids or other extrachromosomal elements, the cells are selectively killed because unstable antitoxins are degraded faster than their cognate stable toxins. Thus, the cells are addicted to the short-lived antitoxins since their de novo synthesis is essential for cell survival.
Among the known addiction modules found on the E. coli chromosome (Gotfredsen and Gerdes, Mol Microbiol 29, 1065-76 (1998); Mittenhuber, J Mol Microbiol Biotechnol 1, 295-302 (1999)), the E. coli MazEF system is the first known prokaryotic chromosomal addiction module (Aizenman et al., Proc Natl Acad Sci USA 93, 6059-63 (1996)). The mazEF module consists of two overlapping genes mazE and mazF, located downstream of the relA gene. MazF is a stable toxin, whereas MazE is a labile antitoxin, which is readily degraded in vivo by an ATP-dependent ClpPA serine protease (Aizenman et al., Proc Natl Acad Sci USA 93, 6059-63 (1996)). mazEF expression is negatively regulated by guanosine 3′,5′-bispyrophosphate (ppGpp) synthesized by RelA under severe amino acid starvation (Aizenman et al., Proc Natl Acad Sci USA 93, 6059-63 (1996)). Moreover, mazEF-mediated cell death can be triggered by several antibiotics, including rifampicin, chloramphenicol and spectinomycin (Sat et al., J Bacteriol 183, 2041-5 (2001)). Results from in vivo experiments using E. coli cells have suggested that MazF inhibits both protein synthesis and DNA replication (Pedersen et al., Mol Microbiol 45, 501-10 (2002)). Thymineless death has recently been reported to be mediated by the mazEF module (B. Sat, M. Reches, H. Engelberg-Kulka, J Bacteriol 185, 1803-7 (2003)).
In E. coli, some extrachromosomal elements are known to contain addiction modules and cause bacterial programmed cell death via the so-called postsegregational killing effect. The best studied extrachromosomal addiction modules include the phd-doc system on bacteriophage P1 (Lehnherr et al. (1993) J Mol Biol 233, 414-428; Gazit and Sauer. (1999) J Biol Chem 274, 16813-16818; Magnuson et al. (1996) J Biol Chem 271, 18705-18710; Lehnherr and Yarmolinsky. (1995) Proc Natl Acad Sci USA 92, 32743277), the ccdA-ccdB system on factor F (Tam and Kline. (1989) J Bacteriol 171, 2353-2360; Bahassi et al. (1999) J Biol Chem 274, 10936-10944; Afif et al. (2001) Mol Microbiol 41, 73-82; Dao-Thi et al. (2002) J Biol Chem 277, 3733-3742), the kis-kid system on plasmid R1 (Ruiz-Echevarria et al. (1991) Mol Microbiol 5, 2685-2693; Hargreaves et al. (2002) Structure (Camb) 10, 1425-1433; Ruiz-Echevarria et al. (1995) J Mol Biol 247, 568-577; Santos-Sierra et al. (2003) Plasmid 50, 120-130), and the pemI-pemK system on plasmid R100 (Tsuchimoto et al. (1992) J Bacteriol 174, 42054211; Tsuchimoto et al. (1988) J Bacteriol 170, 1461-1466; Tsuchimoto and Ohtsubo. (1993) Mol Gen Genet. 237, 81-88; Tsuchimoto and Ohtsubo. (1989) Mol Gen Genet. 215, 463-468). Interestingly, the E. coli chromosome also contains several addiction module systems, such as the relBE system (Gotfredsen and Gerdes. (1998) Mol Microbiol 29, 1065-1076; Christensen et al. (2001) Proc Natl Acad Sci USA 98, 14328-14333; Christensen and Gerdes. (2003) Mol Microbiol 48, 1389-1400; Pedersen et al. (2003) Cell 112, 131-140), the mazEF system (Aizenman et al. (1996) Proc Natl Acad Sci USA 93, 6059-6063; Marianovsky et al. (2001) J Biol Chem 276, 5975-5984; Kamada et al. (2003) Mol Cell 11, 875-884; Zhang et al. (2003) J Biol Chem 278, 32300-32306) and the chpB system (Santos Sierra et al. (1998) FEMS Microbiol Lett 168, 51-58; Masuda et al. (1993) J Bacteriol 175, 6850-6856; Christensen et al. (2003) J Mol Biol 332, 809-819).
The cellular effects of toxins associated with addiction modules have been studied quite extensively. CcdB, the toxin in the ccdA-ccdB system, interacts with DNA gyrase to block DNA replication (Bahassi et al. (1999) supra; Kampranis et al. (1999) J Mol Biol 293, 733-744), and RelE, the toxin in the relBE system cleaves mRNA in the ribosome A site with high codon-specificity, but is not able to degrade free RNA (Pedersen et al. (2003) supra). It was recently demonstrated, however, that the A-site mRNA cleavage can occur in the absence of RelE (Hayes and Sauer. (2003) Mol Cell 12, 903-911). The exact mechanism of the A-site mRNA cleavage, therefore, is still unknown. It has been proposed that MazF (ChpAK), the toxin encoded by the mazEF system, and ChpBK, the toxin encoded by chpB system, inhibit translation by a mechanism very similar to that of RelE in a ribosome-dependent and codon-specific manner (Christensen et al. (2003) supra). The present inventors have, however, recently demonstrated that MazF is a sequence-specific endoribonuclease functional only for single-stranded RNA, which preferentially cleaves mRNAs at the ACA sequence in a manner independent of ribosomes and codons, and is, therefore, functionally distinct from RelE (Zhang et al. (2003) Mol Cell 12, 913-923).
The pemI-pemK system and the kis-kid system are involved in the stable maintenance of two closely related incFII low-copy plasmids, plasmid R100 (Tsuchimoto et al. (1992) supra; Tsuchimoto et al. (1988) supra) and plasmid R1 (Ruiz-Echevarria et al. (1991) supra; Bravo et al. (1987) Mol Gen Genet 210, 101-110), respectively. These two systems are now known to be identical (Engelberg-Kulka and Glaser. (1999) supra). It has been demonstrated that Kid (PemK) inhibits ColE1 plasmid replication acting at the initiation of DNA synthesis, but does not inhibit P4 DNA replication in vitro (Ruiz-Echevarria et al. (1995) supra). To date, there is no evidence that Kid (PemK) inhibits chromosomal DNA replication. Toxin Kid (PemK) and antidote Kis (PemI) not only function in bacteria, but also function efficiently in a wide range of eukaryotes. Kid (PemK) inhibits proliferation in yeast, Xenopus laevis and human cells, wherein Kis (PemI) abrogates this inhibition (de la Cueva-Mendez et al. (2003) Embo J 22, 246-251). It has also been demonstrated that Kid (PemK) triggers apoptosis in human cells (de la Cueva-Mendez et al. (2003) supra). These results suggest that there is a common target for Kid (PemK) in both prokaryotes and eukaryotes.
The citation of references herein shall not be construed as an admission that such is prior art to the present invention.
Other features and advantages of the invention will be apparent from the detailed description, the drawings, and the claims.